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Interpretive Summary: Plants are subject to a large number of diseases, and many of these are caused by viruses. Identifying new viruses that are causing diseases can be very difficult. Most plant virus tests require significant amounts of research before the proper assay can be developed. Very few assays are capable of detecting novel or emerging plant viruses, and the few assays that are useful for discovery provide very little information about these novel viruses. This paper describes a new approach for virus discovery using high throughput technology and the genetics of viruses. Plants often respond to viruses by chewing up the genetic material of the invader. These chewed up nucleic acids end up in a pool of degraded products called “small RNAs”. The novel approach determines the sequence of these “small RNAs” and uses computer technology to reassemble the small bits into the near complete viral genome. This technique was used to identify a novel virus of citrus called Citrus leprosis virus cytoplasmic nuclear type (CiLV-N), a significant problem for citrus production in Brazil and Mexico, and a potential threat to US citrus production. This is the first report of the full genome sequence of CiLV-N, as well as the first sequence of a this class of virus from citrus.

Technical Abstract: Citrus leprosis is a difficult viral disease causing significant damage to citrus fruit in South America and Central America. The disease is marked by dramatic lesions on fruit, leaves and stems resulting in unmarketable product. Citrus leprosis virus cytoplasmic type (CiLV-C) was detected in states of Chipas and Tobasco in Mexico, 2005-06. In 2011-12, CiLV affected Hamlin and Valencia oranges from Mexico were sent to the USDA-APHIS-PPQ-CPHST, Beltsville, MD, USA for testing. Leprosis samples from state of Queretaro failed to react with CiLV-C and CiLV-C type 2 (CiLV-C2) specific antisera in ELISA tests. Furthermore, in RT-PCR CiLV-C and -C2 specific primers also failed to produce amplicons. However, transmission electron microscopy of the infected leaves showed bullet shaped virions in the nuclei and cytoplasm similar to that reported for CiLV nuclear type (CiLV-N). To determine genome sequence of CiLV-N, total RNA from two leprosis samples were sent for small RNA (sRNA) sequencing (Fasteris, S.A., Switzerland) utilizing the Illumina GA IIX platform. After subtracting the sequences of the citrus genome and known viruses, the remaining sequence pools were assembled by tiling using the Velvet and Oases sequence programs. After eliminating the overlapping sequences, the assembled sRNA (20-25 nt) contigs identified the partial genome sequence of CiLV-N. The contigs were recognized utilizing NCBI non-redundant protein database with blastx. The complete genome sequence and structure of CiLV-N was then determined. The CiLV-N genome is composed of RNA1 (~6.4 Kb) and RNA2 (~6.0 Kb) and showed 92-95% nucleotide sequence identity with Orchid fleck virus (OFV) infecting Cymbidium species. However, RNA-1 and -2 sequences of the CiLV-N showed 96-98 percent amino acid identities with OFV sequences and occupied same clade in the phylogenetic tree, suggests that the virus may be a strain of OFV.